The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
In the specific binding assays, such as e.g. immunoassays, DNA hybridization assays, receptor-binding assays, and cellular binding assays, generally the analytes to be measured are present at very low concentrations. Therefore various labelling compounds have been developed that allow the labelled reactant to be detected and quantitated at a high sensitivity. Originally, radioisotopic labels were solely used, but recently, because of the limitations of those labels (such as limited shelf life, safety, health and waste problems and so on) a number of non-radioactive alternatives have been introduced. Fluorometry is a versatile label technology found wide applications in a number of analytical assays based on bioaffinity reactions, such as in situ hybridization, immunohisto and cytochemistry, and also in quantitative fluoroimmunoassays. Although theoretically very sensitive, fluorometric determination is very vulnerable to interferences caused by background sources (Soini, E. and Hemmila, I., 1979, Clin Chem 25; 353 ).
Application of delayed detection of fluorescence excited with a short pulse is a frequently used system to avoid background problems by taking advantage of the temporal separation of specific fluorescence from unspecific background interference of shorter decay-time (Hemmila, I., 1991, Applications of Fluorescence in Immunoassays, Wiley, New York). The system requires, however, a photoluminescent (referred in the context of this text simply as luminescent) label having an excited state lifetime clearly longer than the average background noise. Therefore, the research is focused in developing luminescent lanthanide chelates, which because of the electronic structure of the ions, have unique luminescence properties including exceptionally long decay times ranging from microseconds to milliseconds. A system based on two different chelates, one optimized for labelling and the other for fluorometric determination, DELFIA.RTM. (trademark of Wallac) (U.S. Pat. No. 4,565,790, U.S. Pat. No. 4,808,541) has found established applications particularly in clinical diagnostics. Because of the dissociation step required, this system does not, however, suit for analysis where spatial information is also needed, e.g. for in situ immunofluorescence staining, or in situ hybridizations, neither for multianalyte assays based of distinct solid-phases. Therefore, a number of attempts have been made to develop new highly luminescent chelate labels suitable for those types of time-resolved fluorometric applications. These include e.g. stable chelates composed of derivatives of pyridines (U.S. Pat. No. 4,920,195, U.S. Pat. No. 4,801,722, U.S. Pat. No. 4,761,481, PCT WO FI-91/00373), bipyridines (U.S. Pat. No. 5,216,134), terpyridines (U.S. Pat. No. 4,859,777, U.S. Pat. No. 5,202,423) or various phenolic compounds (U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191) as the energy mediating groups and polycarboxylic acids as chelating parts. In addition various dicarboxylate derivatives (U.S. Pat. No. 5,032,677, U.S. Pat. No. 5,055,578, U.S. Pat. No. 4,772,563), macrocyclic cryptates (U.S. Pat. No. 4,927,923, PCT WO 93/5049, EP-A 493,745) and macrocyclic Schiff bases (EP-A 369,000) have been patented. The problem still existing with all these compounds relates to the relatively low quantum yield, generally decays apart from the optimum (100%) level. Regardless of employment of very stable chelates, such as the cage-type cryptates or nine-dentate terpyridine derivatives, there still remains problems of non-radiative quenching, especially profound with the positively charged cryptates caused by anionic compounds derived from biological samples (PCT WO 92/1224), but even the nine-dentate chelates synthesized so far do not reach 100% quantum yield. It is well known in the literature, that the high energy vibrations of O--H and C--H stretching (vibrational manifold with harmonic overtones) play a leading role in the non-radiative energy transfer processes in the lanthanide chelates (Sinha, A. P. B., 1971, In Rao, C. N. and Ferrano J. R. (ed.), Spectroscopy in Inorganic Chemistry, Horrocks, W. DeW. and Sudnick, D. R., 1981, Acc Chem Res 14; 384-92).